EP2267024B1 - Herstellung von rekombinantem Il-18 bindendem Protein - Google Patents

Herstellung von rekombinantem Il-18 bindendem Protein Download PDF

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EP2267024B1
EP2267024B1 EP10184728A EP10184728A EP2267024B1 EP 2267024 B1 EP2267024 B1 EP 2267024B1 EP 10184728 A EP10184728 A EP 10184728A EP 10184728 A EP10184728 A EP 10184728A EP 2267024 B1 EP2267024 B1 EP 2267024B1
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perfusion
cells
run
protein
cell
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EP2267024A1 (de
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Urs Wieber
Thierry Ziegler
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Ares Trading SA
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
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    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P9/00—Drugs for disorders of the cardiovascular system
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Definitions

  • the present invention is in the field of protein production. More specifically, it relates to a process for the production of recombinant IL-18 binding protein (IL-18BP).
  • the invention further relates to an IL-18BP composition characterized by a specific glycosylation profile.
  • Proteins have become commercially important as drugs that are also generally called "biologicals".
  • biologicals One of the greatest challenges is the development of cost effective and efficient processes for the production of recombinant proteins on a commercial scale.
  • fed-batch or perfusion production mode is mainly dictated by the biology of the clone and the property of the product, and is done on a case-by-case basis during the course of the development of a new drug product (Kadouri and Spier 1997).
  • one of the culture systems of choice is stationary packed-bed bioreactor in which cells are immobilized onto solid carriers. This system is easy to operate and with appropriate carriers and culture conditions very high cell density (of ⁇ 10 7 -10 8 cell ⁇ m -1 ) can be achieved.
  • the optimal stationary packed-bed bioreactor process should operate with a perfusion rate as low as possible without compromising on quantity and quality of the product.
  • glycosylation is usually recognized as an important function in the solubility, immunogenicity, and pharmacokinetic properties of human glycoproteins and those are key parameters in the safety and clinical efficacy of a product (Goochee et al. 1991).
  • glycosylation affects folding and secretion of many glycoproteins, as well as their plasma half-life, thus having an important impact on in vivo biology and activity of glycosylated proteins.
  • glycosylation of a protein refers to the formation of the sugar-amino acid linkage. Glycosylation is a crucial event in the biosynthesis of the carbohydrate units of (secreted) glycoproteins. It sets into motion a complex series of posttranslational enzymatic steps that lead to the formation of a host of protein-bound oligosaccharides with diverse biological functions.
  • Mammalian glycoproteins commonly contain three types of constituent glycans; the N-linked glycans which are attached to asparagine via an N-acetylglucosamine (GlcNAc) residue in an Asn-Xxx-(Ser, Thr) motif, where Xxx can be any amino acid except proline, those attached to serine or threonine, referred to as O-linked glycans and the carbohydrate components of glycosylphosphatidylinositol.
  • the antennae of mature glycans usually consist of one or more N-acetyllactosamine units with the chains terminating in either sialic acid or ⁇ -linked galactose.
  • Fucose is frequently found attached to the asparagine-linked GlcNAc residue and often, additionally on the antennae.
  • Other common modifications to the basic structure include a GlcNAc residue attached to the 4-position of the core branching mannose residue, referred to as a "bisecting" GlcNAc residue and sulphate groups which can be found in a variety of locations, both on the core and the antennae.
  • One such glycosylated protein of therapeutic interest is interleukin-18 binding protein.
  • Interleukin-18 binding protein is a naturally occurring soluble protein that was initially affinity purified, on an IL-18 column, from urine (Novick et al. 1999). IL-18BP abolishes IL-18 induction of IFN- ⁇ and IL-18 activation of NF- ⁇ B in vitro. In addition, IL-18BP inhibits induction of IFN- ⁇ in mice injected with LPS.
  • the IL-18BP gene was localized to the human chromosome 11, and no exon coding for a transmembrane domain could be found in the 8.3 kb genomic sequence comprising the IL-18BP gene.
  • Four isoforms of IL-18BP generated by alternative mRNA splicing have been identified in humans so far. They were designated IL-18BP a, b, c, and d, all sharing the same N-terminus and differing in the C-terminus (Novick et al 1999). These isoforms vary in their ability to bind IL-18 (Kim et al. 2000).
  • isoforms a and c are known to have a neutralizing capacity for IL-18.
  • the most abundant IL-18BP isoform, isoform a exhibits a high affinity for IL-18 with a rapid on-rate and a slow off-rate, and a dissociation constant (Kd) of approximately 0.4 nM (Kim et al. 2000).
  • IL-18BP belongs to the immunoglobulin superfamily.
  • IL-18BP has been suggested as a therapeutic protein in a number of diseases and disorders, such as psoriasis, Crohn's Disease, rheumatoid arthritis, psoriatic arthritis, liver injury, sepsis, atherosclerosis, ischemic heart diseases, allergies, etc., see e.g. WO9909063 , WO0107480 , WO0162285 , WO0185201 , WO02060479 , WO02096456 , WO03080104 , WO02092008 , WO02101049 , WO03013577 .
  • diseases and disorders such as psoriasis, Crohn's Disease, rheumatoid arthritis, psoriatic arthritis, liver injury, sepsis, atherosclerosis, ischemic heart diseases, allergies, etc., see e.g. WO9909063 , WO0107480 , WO0162285 , WO0185
  • the prior art does not describe a process for the production of recombinant IL-18BP in CHO cells, nor IL-18BP compositions characterized by a specific glycosylation profile.
  • the present invention relates to the development of a process for producing recombinant Interleukin-18 binding protein (IL-18BP) in Chinese Hamster Ovary (CHO) cells in a bioreactor under serum-free culture conditions comprising a cell propagation phase at 37°C and a production phase at 29°C.
  • IL-18BP Interleukin-18 binding protein
  • An efficient fed-batch process has been developed for the production of IL-18BP in CHO cells, under serum-free culture conditions, comprising:
  • the present invention is based on the development of efficient processes for the production of recombinant IL-18BP in a bioreactor under serum-free cell culture conditions. Therefore, the present invention relates to a process for producing recombinant Interleukin-18 binding protein (IL-18BP) in CHO cells in a bioreactor under serum-free culture conditions comprising a cell propagation phase at 37°C and a production phase at 29 °C.
  • IL-18BP Interleukin-18 binding protein
  • Any temperature between approximately 29 to approximately 34°C will be suitable in the frame of the present disclosure such as e.g. 28, 28.5, 29, 29.5, 30, 30.5, 31, 31.5, 32, 32.5, 33, 33.5, 34, 34.5, 35 °C.
  • a perfusion process for producing recombinant Interleukin-18 binding protein (IL-18BP) in mammalian cells in a bioreactor under serum-free culture conditions comprising:
  • the perfusion rate of production phase may be e.g. at about 86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66 or 65, or 64 % of the initial perfusion rate in step (a).
  • the temperature may vary within certain limits, so as to be e.g. about 37.5 °C in step (a) or 34-33 °C in step (b) or 32-33 °C in step (c).
  • the Dissolved Oxygen concentration (DO) of a cell culture is maintained at about 50 to 70% of air saturation, and pH values vary between 6.5 and 7.5, preferably around 7.0.
  • Bioreactor refers to an apparatus or closed container that is used for generating biomolecules such as secreted proteins using the synthetic or chemical conversion capacity of a cell.
  • Bioreactors include classical fermenters and cell culture perfusion systems. Bioreactors allow controlling various parameters during the cell culture process such as, e.g., the circulation loop flow, the temperature, the overpressure and/or the medium perfusion rate.
  • serum-free medium refers to any medium that is free from components derived from animal serum such as e.g. fetal calf serum.
  • examples for commercially available serum-free media that can be used in accordance with the present invention include, e.g., SFM 90 (JRH, 67350), SFM 90.1 (JRH, 67350), Supmed300 or Supmed300 modified (JRH, 67350), DMEM (Gibco, 7490571), DMEM/F12 (Gibco, 99.5043), SFM CHO 3a (BioWhittaker), CHO PFM (Sigma, C6970), ProCHO 5, EX-CELL media such as EX-CELL 302 (JRH, Catalogue No.
  • HyQ PF CHO HyClone, Catalogue No. SH30220.01
  • HyQ SFX CHO HyClone, Catalogue No. SH30187.01
  • HyQ CDM4CHO HyClone, Catalogue No. SH30558.01
  • IS CHO-CD Irvine Scientific, Catalogue No. #91119
  • IS CHO-V Irvine Scientific, Catalogue No. #9197
  • the composition of SFM 90, SFM 90.1, SupMed300, DMEM, DMEM/F12, SFM CHO 3 a and CHP PFM is shown in Table I below.
  • the serum-free medium may preferably be a chemically defined medium, i.e., a medium prepared from purified ingredients and therefore whose exact composition is known. Specifically, chemically defined media do neither contain animal derived components nor undefined hydrolysates.
  • the perfusion rate of step (a) has a dilution rate in the range of about 2 to 3 wd, preferably about 2.5 vvd.
  • the perfusion rate of step (a) may be e.g. about 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0 or 3.1 wd.
  • the length of the cell propagation phase of step (a), the production phase I of step (b) and the production phase II of step (c) will be readily determined by the person skilled in the art on the basis of parameters such as the initial cell seed, the type of cells used, the daily measured glucose consumption rate, the dilution rate, and process time.
  • the cells are associated to carriers in the bioreactor, and that production phase I of step (b) is started at a glucose consumption rate of about 250 to 350 g of Glucose per kilogram of carrier.
  • the carrier that may be used in accordance with the processes of present invention may e.g. be a microcarrier.
  • Microcarriers are small solid particles on which cells may be grown in suspension culture. Cells are capable of adhering and propagating on the surface of microcarriers. Typically, microcarriers consist of beads, the diameter of which is comprised between 90 ⁇ m and 300 ⁇ m. Microcarriers can be made of various materials that have proven successful for cell attachment and propagation such as, e.g., glass, polystyrene, polyethylene, dextran, gelatin and cellulose.
  • microcarriers may be coated with a material promoting cell attachment and growth such as, e.g., e.g., N,N-diethylaminoethyl, glass, collagen or recombinant proteins.
  • a material promoting cell attachment and growth such as, e.g., e.g., N,N-diethylaminoethyl, glass, collagen or recombinant proteins.
  • Both macroporous and non-porous microcarriers do exist. Macroporous surfaces give the cells easy access to the interior of the microcarrier after inoculation, and once inside of the microcarrier, the cells are protected from the shear forces generated by mechanical agitation and aeration in the bioreactor.
  • a further solid carrier that may be used in accordance with the present invention may e.g. be a Fibra-Cel ® disk.
  • Fibra-Cel ® disks are disks of 6 mm in diameter that are composed of polyester non-woven fiber bonded to a sheet of polypropylene mesh (see, e.g., U.S. patent No. 5,266,476 and world wide web pages nbsc.com/products/miscellaneous/fibracel/ and nbsc.com/support/faqs/#fibra).
  • Fibra-Cel ® disks are usually treated electrostatically to facilitate suspension cells adhering to the disks and becoming trapped in the fiber system, where they remain throughout the cultivation process. Cell density and productivity achieved with cells grown on Fibra-Cel ® disks can be up to ten times higher than with cells growing on microcarriers.
  • the cells expressing IL-18BP may be any mammalian cell, including animal or human cells, such as e.g. 3T3 cells, COS cells, human osteosarcoma cells, MRC-5 cells, BHK cells, VERO cells, CHO cells, rCHO-tPA cells, rCHO - Hep B Surface Antigen cells, CHO-S cells, HEK 293 cells, rHEK 293 cells, rC127 - Hep B Surface Antigen cells, Normal Human fibroblast cells, Stroma cells, Hepatocytes cells and PER.C6 cells.
  • animal or human cells such as e.g. 3T3 cells, COS cells, human osteosarcoma cells, MRC-5 cells, BHK cells, VERO cells, CHO cells, rCHO-tPA cells, rCHO - Hep B Surface Antigen cells, CHO-S cells, HEK 293 cells, rHEK 293 cells, rC127 - Hep B Surface Antigen cells, Normal Human fibroblast
  • Chinese Hamster Ovary (CHO) cells for expression of IL-18BP are used in a process according to the invention.
  • the processes for production of IL-18BP of the invention preferably further comprises a step of collecting the cell culture supernatant (harvest).
  • the processes further comprise one or more steps of purifying IL-18BP.
  • Any suitable method may be used for the purification of IL-18BP, such as e.g. the purification processes described for IL-18BP in WO 2006/003134 or WO 2005/049649 .
  • the purified IL-18PB product may then preferably be formulated into a pharmaceutical composition.
  • the disclosure also relates to an IL-18BP composition characterized by a sialylation profile comprising about 15 to about 25%, preferably about 19 to about 21% of unsialylated N-glycans, about 15 to about 30%, preferably about 20 to about 25% of mono-sialylated glycans, about 35 to about 55%, preferably about 39% to about 44% of di-sialylated N-glycans, about 5 to about 15%, preferably about 7 to about 10% of tri-sialylated N-glcyans and about 1 to about 5%, preferably about 2% to about 3% of tetra-sialylated N-glycans.
  • a sialylation profile comprising about 15 to about 25%, preferably about 19 to about 21% of unsialylated N-glycans, about 15 to about 30%, preferably about 20 to about 25% of mono-sialylated glycans, about 35 to about 55%, preferably about 3
  • Such an IL-18BP composition is preferably obtained by the production processes of the invention.
  • the present invention relates to a fed-batch process for producing recombinant Interleukin-18 binding protein (IL-18BP) in CHO cells in a bioreactor under serum-free culture conditions comprising the steps of:
  • phase (a), optionally (b), and (c) can be readily determined by the person skilled in the art.
  • Phase (a) will continue until an adequate number of cells has been generated, such as e.g. in the range of 10 5 to 10 6 .
  • Phase (b) will preferably be short, as it is a transient phase serving at adaptation of the cells to lower temperatures.
  • the total cell density in the production phase ranges between 4 to 8 x 10 6 cells per ml per day, preferably over at least 10 days of cell culture.
  • the viability ranges between 100 and 80 %, preferably over at least 10 days of cell culture.
  • the protein productivity is higher than about 150 mg or about 250 mg or about 350 per I per day.
  • Mammalian cells to be used in the frame of the process of the present invention are Chinese Hamster Ovary (CHO) cells.
  • the process preferably further comprises the steps of collecting the cell culture supernatant and/or purifying IL-18BP and/or formulating the IL-18BP into a pharmaceutical composition.
  • Th disclosure also relates to an IL-18BP composition having a glycoform profile comprising about 0 to about 15 % of basic glycoforms, about 15 to about 30 % of less acidic glycoforms, about 45 to about 65 % of acidic glycoforms, and about 10 to about 25 % of highly acidic glycoforms.
  • the glyocoform profile is characterized by way of capillary zone electrophoresis (CZE), indicating the extent of basic, less acidic, acidic and highly acidic glycoforms of IL-18BP.
  • CZE capillary zone electrophoresis
  • the hypothetical charge number Z is a parameter calculated according to a known formula (Hermentin et al., 1996, Gervais et al., 2003, see also the examples below), and characterizes the extent of sialylation of glycoproteins.
  • the IL-18BP composition is characterized by a Z-number ranging between about 130 to about 160, preferably between about 140 and 155, more preferably between about 145 and about 150.
  • Preferred Z-numbers for IL-18BP are e.g. 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, or 156.
  • the IL-18BP compositions may be produced by any process, such as e.g. perfusion processes operated with different parameter settings than described herein, e.g. at higher perfusion rates, the IL-18BP compositions are preferably produced in the fed-batch process of the present invention.
  • IL-18BP to be produced may be any IL-18BP from any species. Preferably, it is human IL-18BP.
  • IL-18BP is a soluble, secreted protein, it is released into the cell culture supernatant, either by means of its natural signal peptide, or by means of a heterologous signal peptide, i.e. a signal peptide derived from another secreted protein which may be more efficient in the particular expression system used, such as e.g. the GH signal peptide.
  • IL-18 binding protein is used herein synonymously with "IL-18BP". This term relates IL-18 binding proteins such as the ones defined in WO 99/09063 or in Novick et al., 1999 .
  • IL-18BP includes splice variants and/or isoforms of IL-18 binding proteins, as the ones defined in Kim et al., 2000, in particular human isoforms a and c of IL-18BP.
  • IL-18PB further includes muteins, functional derivatives, active fractions, fused proteins, circularly permutated proteins and salts of IL-18BP as defined in WO 99/09063 .
  • muteins refers to analogs of an IL-18BP, or analogs of a viral IL-18BP, in which one or more of the amino acid residues of a natural IL-18BP or viral IL-18BP are replaced by different amino acid residues, or are deleted, or one or more amino acid residues are added to the natural sequence of an IL-18BP, or a viral IL-18BP, without changing considerably the activity of the resulting products as compared with the wild type IL-18BP or viral IL-18BP.
  • muteins are prepared by known synthesis and/or by site-directed mutagenesis techniques, or any other known technique suitable therefor.
  • Muteins include proteins encoded by a nucleic acid, such as DNA or RNA, which hybridizes to DNA or RNA, which encodes an IL-18BP or encodes a viral IL-18BP ( W09909063 ) under stringent conditions.
  • stringent conditions refers to hybridization and subsequent washing conditions, which those of ordinary skill in the art conventionally refer to as “stringent”. See Ausubel et al., Current Protocols in Molecular Biology, supra, Interscience, N.Y., ⁇ 6.3 and 6.4 (1987, 1992 ).
  • examples of stringent conditions include washing conditions 12-20°C below the calculated Tm of the hybrid under study in, e.g., 2 x SSC and 0.5% SDS for 5 minutes, 2 x SSC and 0.1% SDS for 15 minutes; 0.1 x SSC and 0.5% SDS at 37°C for 30-60 minutes and then, a 0.1 x SSC and 0.5% SDS at 68°C for 30-60 minutes.
  • stringency conditions also depend on the length of the DNA sequences, oligonucleotide probes (such as 10-40 bases) or mixed oligonucleotide probes. If mixed probes are used, it is preferable to use tetramethyl ammonium chloride (TMAC) instead of SSC. See Ausubel, supra.
  • Identity reflects a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, determined by comparing the sequences. In general, identity refers to an exact nucleotide to nucleotide or amino acid to amino acid correspondence of the two polynucleotides or two polypeptide sequences, respectively, over the length of the sequences being compared.
  • a "% identity" may be determined.
  • the two sequences to be compared are aligned to give a maximum correlation between the sequences. This may include inserting "gaps" in either one or both sequences, to enhance the degree of alignment.
  • a % identity may be determined over the whole length of each of the sequences being compared (so-called global alignment), that is particularly suitable for sequences of the same or very similar length, or over shorter, defined lengths (so-called local alignment), that is more suitable for sequences of unequal length.
  • Any such mutein preferably has a sequence of amino acids sufficiently duplicative of that of an IL-18BP, or sufficiently duplicative of a viral IL-18BP, such as to have substantially similar activity to IL-18BP.
  • One activity of IL-18BP is its capability of binding IL-18.
  • the mutein can be used in the purification of IL-18, such as by means of affinity chromatography, and thus can be considered to have substantially similar activity to IL-18BP.
  • any given mutein has substantially the same activity as IL-18BP by means of routine experimentation comprising subjecting such a mutein, e.g., to a simple sandwich competition assay to determine whether or not it binds to an appropriately labeled IL-18, such as radioimmunoassay or ELISA assay.
  • a simple sandwich competition assay to determine whether or not it binds to an appropriately labeled IL-18, such as radioimmunoassay or ELISA assay.
  • any such mutein has at least 40% identity or homology with the sequence of either an IL-18BP or a virally encoded IL-18BP homologue, as defined in WO 99/09063 . More preferably, it has at least 50%, at least 60%, at least 70%, at least 80% or, most preferably, at least 90% identity or homology thereto.
  • Muteins of IL-18BP polypeptides or muteins of viral IL-18BPs, which can be used, or nucleic acid coding therefor, include a finite set of substantially corresponding sequences as substitution peptides or polynucleotides which can be routinely obtained by one of ordinary skill in the art, without undue experimentation, based on the teachings and guidance presented herein.
  • muteins are what are known as "conservative" substitutions.
  • Conservative amino acid substitutions of IL-18BP polypeptides or proteins or viral IL-18BPs may include synonymous amino acids within a group which have sufficiently similar physicochemical properties that substitution between members of the group will preserve the biological function of the molecule (Grantham, 1974). It is clear that insertions and deletions of amino acids may also be made in the above-defined sequences without altering their function, particularly if the insertions or deletions only involve a few amino acids, e.g., under thirty, and preferably under ten, and do not remove or displace amino acids which are critical to a functional conformation, e.g., cysteine residues. Proteins and muteins produced by such deletions and/or insertions come within the purview of the present disclosure.
  • the synonymous amino acid groups are those defined in Table 4. More preferably, the synonymous amino acid groups are those defined in Table 5; and most preferably the synonymous amino acid groups are those defined in Table 6. TABLE I Preferred Groups of Synonymous Amino Acids Amino Acid Synonymous Group Ser Ser, Thr, Gly, Asn Arg Arg, Gln, Lys, Glu, His Leu Ile, Phe, Tyr, Met, Val, Leu Pro Gly, Ala, Thr, Pro Thr Pro, Ser, Ala, Gly, His, Gln, Thr Ala Gly, Thr, Pro, Ala Val Met, Tyr, Phe, Ile, Leu, Val Gly Ala, Thr, Pro, Ser, Gly Ile Met, Tyr, Phe, Val, Leu, Ile Phe Trp, Met, Tyr, Ile, Val, Leu, Phe Tyr Trp, Met, Phe, Ile, Val, Leu, Tyr Cys Ser, Thr, Cys His Glu, Lys, G
  • Examples of production of amino acid substitutions in proteins which can be used for obtaining muteins of IL-18BP polypeptides or proteins, or muteins of viral IL-18BPs include any known method steps, such as presented in US patents 4,959,314 , 4,588,585 and 4,737,462, to Mark et al ; 5,118,943 to Koths et al. , 4,965,195 to Namen et al ; 4,879,111 to Chong et al ; and 5,017,691 to Lee et al ; and lysine substituted proteins presented in US patent No. 4,904,584 (Shaw et al ).
  • fused protein refers to a polypeptide comprising an IL-18BP, or a viral IL-18BP, or a mutein or fragment thereof, fused with another protein, which, e.g., has an extended residence time in body fluids.
  • An IL-18BP or a viral IL-18BP may thus be fused to another protein, polypeptide or the like, e.g., an immunoglobulin or a fragment thereof.
  • “Functional derivatives” as used herein cover derivatives of IL-18BPs or a viral IL-18BP, and their muteins and fused proteins, which may be prepared from the functional groups which occur as side chains on the residues or the N- or C-terminal groups, by means known in the art, and are included in the disclosure as long as they remain pharmaceutically acceptable, i.e. they do not destroy the activity of the protein which is substantially similar to the activity of IL-18BP, or viral lL-1 8BPs, and do not confer toxic properties on compositions containing it.
  • derivatives may, for example, include polyethylene glycol side-chains, which may mask antigenic sites and extend the residence of an IL-18BP or a viral IL-18BP in body fluids.
  • Other derivatives include aliphatic esters of the carboxyl groups, amides of the carboxyl groups by reaction with ammonia or with primary or secondary amines, N-acyl derivatives of free amino groups of the amino acid residues formed with acyl moieties (e.g. alkanoyl or carbocyclic aroyl groups) or O-acyl derivatives of free hydroxyl groups (for example that of seryl or threonyl residues) formed with acyl moieties.
  • acyl moieties e.g. alkanoyl or carbocyclic aroyl groups
  • O-acyl derivatives of free hydroxyl groups for example that of seryl or threonyl residues
  • active fractions of an IL-18BP, or a viral IL-18BP, muteins and fused proteins covers any fragment or precursors of the polypeptide chain of the protein molecule alone or together with associated molecules or residues linked thereto, e.g., sugar or phosphate residues, or aggregates of the protein molecule or the sugar residues by themselves, provided said fraction has substantially similar activity to IL-18BP.
  • salts herein refers to both salts of carboxyl groups and to acid addition salts of amino groups of IL-18 inhibitor molecule, or analogs thereof.
  • Salts of a carboxyl group may be formed by means known in the art and include inorganic salts, for example, sodium, calcium, ammonium, ferric or zinc salts, and the like, and salts with organic bases as those formed, for example, with amines, such as triethanolamine, arginine or lysine, piperidine, procaine and the like.
  • Acid addition salts include, for example, salts with mineral acids, such as, for example, hydrochloric acid or sulfuric acid, and salts with organic acids, such as, for example, acetic acid or oxalic acid.
  • any such salts must retain the biological activity of the IL-18 inhibitor, such as induction of IFN-gamma in blood cells.
  • sequences of IL-18BP and its splice variants/isoforms can be taken from WO99/09063 or from Novick et al., 1999, as well as from Kim et al., 2000.
  • IL-18BP may be conjugated to polymers in order to improve the properties of the protein, such as the stability, half-life, bioavailability, tolerance by the human body, or immunogenicity.
  • IL18-BP may be linked e.g. to Polyethlyenglycol (PEG). PEGylation may be carried out by known methods, described in WO 92/13095 , for example.
  • a fusion protein of IL-18BP may e.g. comprise an immunoglobulin fusion, i.e. the inhibitor of IL-18 is a fused protein comprising all or part of an IL-18 binding protein, which is fused to all or a portion of an immunoglobulin.
  • Methods for making immunoglobulin fusion proteins are well known in the art, such as the ones described in WO 01/03737 , for example.
  • the person skilled in the art will appreciate that the resulting fusion protein substantially retains the biological activity of IL-18BP, such as e.g. the binding to IL-18 which can be measured in in vitro assays described in the prior art such as e.g. WO 99/09063 .
  • the fusion may be direct, or via a short linker peptide which can be as short as 1 to 3 amino acid residues in length or longer, for example, 13 amino acid residues in length.
  • Said linker may be a tripeptide of the sequence E-F-M (Glu-Phe-Met), for example, or a 13-amino acid linker sequence comprising Glu-Phe-Gly-Ala-Gly-Leu-Val-Leu-Gly-Gly-Gln-Phe-Met introduced between the IL-18BP sequence and the immunoglobulin sequence.
  • the resulting fusion protein has improved properties, such as an extended residence time in body fluids (half-life), increased specific activity, increased expression level, or the purification of the fusion protein is facilitated.
  • IL-18BP is fused to the constant region of an Ig molecule, e.g. an Fc portion of an Immunoglobulin.
  • Ig molecule e.g. an Fc portion of an Immunoglobulin.
  • it is fused to heavy chain regions, like the CH2 and CH3 domains, optionally with the hinge region of human IgG1, for example.
  • the Fc part may e.g. be mutated in order to prevent unwanted activities, such as complement binding, binding to Fc receptors, or the like.
  • Fusion proteins comprising IL-18BP and a portion of an immunoglobulin are described in example 11 of WO 99/09063 , for example.
  • Other isoforms of Ig molecules are also suitable for the generation of fusion proteins, such as isoforms IgG 2 or IgG 4 , or other Ig classes, like IgM or IgA, for example.
  • Fusion proteins may be monomeric or multimeric, hetero- or homomultimeric.
  • Further fusion proteins of IL-18BP may be prepared by fusing domains isolated from other proteins allowing the formation or dimers, trimers, etc.
  • Examples for protein sequences allowing the multimerization of the polypeptides are domains isolated from proteins such as hCG ( WO 97/30161 ), collagen X ( WO 04/33486 ), C4BP ( WO 04/20639 ), Erb proteins ( WO 98/02540 ), or coiled coil peptides ( WO 01/00814 ).
  • IL-18BP produced according to the process of the invention, or the composition as disclosed herein may be intended for therapeutic use, i.e. for administration to patients. If IL-18BP is administered to patients, it is preferably administered systemically, and preferably subcutaneously or intramuscularly, or topically, i.e. locally. Rectal or intrathecal administration may also be suitable, depending on the specific use of IL-18BP.
  • produced IL-18BP may be formulated as a pharmaceutical composition, i.e. together with a pharmaceutically acceptable carrier, excipients or the like.
  • the definition of "pharmaceutically acceptable” is meant to encompass any carrier, which does not interfere with effectiveness of the biological activity of the active ingredient and that is not toxic to the host to which it is administered.
  • the active protein(s) may be formulated in a unit dosage form for injection in vehicles such as saline, dextrose solution, serum albumin and Ringer's solution.
  • the active ingredients of the pharmaceutical composition as disclosed herein can be administered to an individual in a variety of ways.
  • the routes of administration include intradermal, transdermal (e.g. in slow release formulations), intramuscular, intraperitoneal, intravenous, subcutaneous, oral, intracranial, epidural, topical, rectal, and intranasal routes. Any other therapeutically efficacious route of administration can be used, for example absorption through epithelial or endothelial tissues or by gene therapy wherein a DNA molecule encoding the active agent is administered to the patient (e.g. via a vector), which causes the active agent to be expressed and secreted in vivo.
  • the protein(s) can be administered together with other components of biologically active agents such as pharmaceutically acceptable surfactants, excipients, carriers, diluents and vehicles.
  • the active protein(s) can be formulated as a solution, suspension, emulsion or lyophilized powder in association with a pharmaceutically acceptable parenteral vehicle (e.g. water, saline, dextrose solution) and additives that maintain isotonicity (e.g. mannitol) or chemical stability (e.g. preservatives and buffers).
  • a pharmaceutically acceptable parenteral vehicle e.g. water, saline, dextrose solution
  • additives that maintain isotonicity e.g. mannitol
  • chemical stability e.g. preservatives and buffers.
  • the therapeutically effective amounts of the active protein(s) will be a function of many variables, including the type of antagonist, the affinity of the antagonist for IL-18, any residual cytotoxic activity exhibited by the antagonists, the route of administration, the clinical condition of the patient (including the desirability of maintaining a non-toxic level of endogenous IL-18 activity).
  • a “therapeutically effective amount” is such that when administered, the IL-18 inhibitor results in inhibition of the biological activity of IL-18.
  • the dosage administered, as single or multiple doses, to an individual will vary depending upon a variety of factors, including IL-18 inhibitor pharmacokinetic properties, the route of administration, patient conditions and characteristics (sex, age, body weight, health, size), extent of symptoms, concurrent treatments, frequency of treatment and the effect desired. Adjustment and manipulation of established dosage ranges are well within the ability of those skilled in the art, as well as in vitro and in vivo methods of determining the inhibition of IL-18 in an individual.
  • IL-18BP may be used in amounts in the ranges of about 0.001 to 100 mg/kg or about 0.01 to 10 mg/kg or body weight, or about 0. 1 to 5 mg/kg of body weight or about 1 to 3 mg/kg of body weight or about 2 mg/kg of body weight.
  • IL-18BP may be administered daily or every other day or three times per week or once per week, at similar doses, or at doses increasing or decreasing with the time.
  • Second or subsequent administrations can be performed at a dosage which is the same, less than or greater than the initial or previous dose administered to the individual.
  • a second or subsequent administration can be administered during or prior to onset of the disease.
  • IL-18BP may be administered prophylactically or therapeutically to an individual prior to, simultaneously or sequentially with other therapeutic regimens or agents (e.g. multiple drug regimens), in therapeutically effective amounts.
  • IL-18BP produced in accordance with the present invention may be used for preparation of a medicament for treatment and/or prevention of a number of diseases or disorders.
  • diseases or disorders may e.g. be IL-18 mediated disorders.
  • IL-18BP may be used for treatment and/or prevention of psoriasis, psoriatic arthritis, Crohn's Disease, inflammatory bowel disease, rheumatoid arthritis, liver injury such as alcoholic liver cirrhosis, sepsis, atherosclerosis, ischemic heart diseases, allergies, in particular delayed-type hypersensitivity, and closed head injury.
  • Example 1 Perfusion process for the production of recombinant, human IL-18BP from serum-free CHO cell harvest in packed-bed bioreactor
  • This example describes a process based on the high cell density culture of recombinant CHO cells in a packed-bed bioreactor, in which the perfusion rate was adjusted according to cell growth requirements during the growth phase and then pronouncedly reduced during the production phase without compromising process productivity or protein quality.
  • the protein product produced by this process was characterized and turned out to have an advantageous N-glycan-profile.
  • a first-generation process had originally been designed with the aim to rapidly produce material for pre-clinical and early clinical trials.
  • This process was designed with a high perfusion rate of 2.6 wd in order to supply the high cell density (- 2.5 ⁇ 10 7 cell ⁇ ml -1 of packed-bed) with fresh medium during production phase.
  • product degradation was not a concern since the high dilution rate imposed to the culture maintained a low residence time of the product, IL-18BP, in the bioreactor environment.
  • a packed-bed bioreactor (Ducommun et al. 2002a; Ducommun et al. 2002b) with Fibra-Cel ® carrier (Bibby Sterilin, U.K.) was used to cultivate CHO cells (Laboratoires Serono S.A., Corsier-sur-Vevey, Switzerland) that express and secrete IL-18BP in a serum free medium (Sigma C-9486).
  • the bioreactor and packed-bed had a working volume of 15 and 5 litres respectively.
  • the bioreactor was perfused with 2.6 wd (as defined in Table 1) during growth and production phase. This basic perfusion rate is chosen as the reference 100%, stated as run-100.
  • Table 1 Perfusion rates tested during the production phase for run-100, run-75 and run-50. Average of n replicates ( ⁇ 2 standard deviations for run-1 00).
  • medium was perfused at 100% during growth phase at 37°C.
  • the temperature was regulated at 37.0°C during growth phase, and then reduced in two steps down to 32.5°C.
  • the pH was regulated at 7.00 and Dissolved Oxygen concentration (DO) was maintained at 70% of air saturation throughout the culture.
  • GCR Glucose Consumption Rate
  • This stage has been defined as the end of the cell propagation phase at 37°C, and when GCR reached a level of 300 grams of glucose per kilogram of Fibra-Cel ® disks per day, the cultures were switched from 37.0°C to the production mode by lowering the temperature to 33.5°C.
  • the perfusion was kept at 100% (run-100) and the two other sets were performed with a medium perfusion rate of 75% (run-75) and 50% (run-50) of the maximal level, as summarized in Table 1.
  • the temperature was further decreased to 32.5°C at a later stage of the production phase to prevent further cell growth and to promote production.
  • the bars displayed on the figures represent an interval of 4 standard deviations ( ⁇ 2 standard deviation) measured for the 8 replicates ran under the conditions of run-1 00.
  • the produced recombinant IL18BP was quantified with an ELISA test.
  • the quality of the recombinant protein was assessed with an RP-HPLC method, in combination with SDS-PAGE methods (ExcelGel SDS Homogeneous 12.5% (Cat# 80-1261-01, Pharmacia).
  • the gels were stained specifically by Western Blot in order to detect high molecular weight (i.e. dimers, aggregates) or low molecular weight variants of the IL-18BP.
  • Non-specific staining by Silver Stain was also used to assess the relative intensity of the IL-18BP compared to impurities after scanning (Scanner ARCUS 2, Afga) of individual bands in order to determine their relative intensity.
  • N-glycanase E-5006B or E-5006C, Glyko Inc. were specifically cleaved from the IL-18BP by hydrazinolysis (N-glycanase E-5006B or E-5006C, Glyko Inc.), labelled with the 2-Aminobenzamide fluorescent dye (Signal 2-AB labelling kit K404, Glyko Inc.), and separated by a chromatography column before passing through a fluorescent detector.
  • the proportions of N-glycans species could then be determined after integration of the HPLC peaks corresponding to neutral, mono-, di-, tri-, and tetra-sialylated forms.
  • the first attempt was to reduce the perfusion rate from 2.6 vvd to 2.0 vvd and 1.3 vvd during the production phase ( Fig. 1 ) and to follow its effect on cell metabolism, volumetric productivity and product quality.
  • the concentration of glucose and lactate were measured for all three perfusion rates ( Fig. 2A and B ), and the residual glucose level remained above 0.5 g/L.
  • Fig. 3 show the glucose consumption rate (GCR) levels for the three sets of medium perfusion rate tested. These results indicate that a reduced perfusion rate induces a lower GCR of the culture. However, this effect was barely significant and when the medium perfusion rate was reduced by -25% and -50%, the average GCR measured over the 60-day production phase were reduced by -8% and -15% respectively ( Fig. 3 ).
  • Fig. 5 A, B and C show a comparison of the recombinant protein produced (volumetric productivity, total production, and titre) in the reference run-100 and in the runs with reduced medium perfusion.
  • FIG. 5A Another difference observed in Fig. 5A is the decline of the volumetric productivity over time along the production phase. A stable volumetric productivity was observed for run-1 00 and run-75, but in run-50 productivity declined over the duration of the 60-day production phase. More specifically, the productivity level of run-50 was 60% of the reference value at the end of the 60-day production phase.
  • Fig. 5B represents the cumulated amount of product made over the 60-days production phase.
  • Fig. 5C The corresponding titers measured for the different perfusion rates are presented in Fig. 5C , which shows that recombinant protein titers were increased by +25% and +50% respectively as opposed to the control when the perfusion was decreased by -25% and -50%.
  • the recombinant protein was purified to homogeneity at three points of the bioreactor runs (day 20, 40 and 60) in order to verify that the product quality was maintained for each perfusion rate investigated.
  • the conditions of run-50 were considered as a worst case for protein degradation since this run had the lowest perfusion rate and the longest product residence time in the bioreactor.
  • the high level of purity (-100%) of the IL-18BP produced was confirmed by RP-HPLC for run-1 00, run-75 and run-50.
  • HCP-ELISA Host Cell Proteins
  • the optimal medium perfusion rate to be used for the continuous culture of a recombinant CHO cell line in a packed-bed bioreactor made of Fibra-Cel ® disk carriers was determined.
  • a first-generation process had originally been designed with a high perfusion rate, originally operated with a perfusion of 2.6 vvd during production phase in order to supply the high cell density (- 2.5 ⁇ 10 7 cell ⁇ ml -1 of packed-bed) with sufficient fresh medium.
  • the protein quality under reduced perfusion rate conditions was analysed for purity, N-glycan sialylation level, abundance of dimers or aggregates, and showed that the quality of the final drug substance was comparable to that obtained in high perfusion conditions.
  • Glucose starvation is a typical cause of incorrect product sialylation (Goochee and Monica 1990). This effect has been studied for IFN-g produced from CHO cells, and the product glycosylation was found affected under low glucose residual levels below 0.1 g/L (Hayter et al. 1993), (Hooker et al. 1995).
  • the -25% reduction on medium translated directly into a -25% saving on: the powder medium and side ingredients, the pre-filters and sterilizing filters, the sterile bags used for media storage after filtration, and the labour costs associated with medium preparation as fewer medium batches were needed.
  • Example 2 Fed-batch process for the production of recombinant, human IL-18BP from serum-free CHO cell culture
  • the culture set points were: oxygen concentration of 50% air saturation, pH 7.0 and 6.90, temperature of 37.0°C during growth phase and then reduced in two steps down to 29.0°C.
  • the serum-free basal medium Sigma, S-9942 was gradually supplemented with a concentrated feed solution.
  • Fig. 7(A) to (D) Important performance indicators, namely total cell-density, viability, residual glucose concentration, and protein titer were analysed. As shown in Fig. 7(A) to (D) , the final fed-batch process allowed growing up the clone to a maximum total cell-density of approximately 7.0 mioCs/mL and maintaining cell viability above 80% until working day 19, where the 300L culture was harvested, clarified and captured.
  • Fig. 7(C) corresponds to the residual glucose concentration profiles, based on daily off-line measurements. These profiles illustrate the applied feeding strategy. For example, the first bolus addition of 80 g/kg of supernatant occurred in all three batches on working day 4 in order to raise residual glucose concentration up to more than 5.5 g/L.
  • Example 3 Sialylation profile of rhIL-18BP produced in perfusion and the fed-batch process for CHO cell culture
  • a further perfusion process for production of IL-18BP was set up. Perfusion runs were performed with a perfusion rate 2.75 wd in a bioreactor containing a total volume of 160 L (including external column of 40 L) packed with 4.4 kg of Fibracel®-disks, at production temperatures of 33.5 or 32.5°C.
  • the IL-18BP produced in this perfusion process was compared to the material derived from the fed-batch process described in example 2.
  • the post-capture IL-18BP material was analyzed for N-glycanation as described in Example 1 above.
  • the IL-18BP material (“crude harvest material”), either derived from the fed-batch or the perfusion process, was also analyzed by Capillary Zone Electrophoresis (CZE) for differentially glycosylated isoforms ("glycoforms”), according to the protocol described in detail below.
  • CZE Capillary Zone Electrophoresis
  • the isoform distribution was further compared to purified IL-18BP, either derived from fed-batch or the perfusion process.
  • the purification steps included metal ion affinity chromatography (on Chelating Sepharose FF), hydrophobic charge-induction chromatography (on Mep Hypercel), ion exchange chromatography (on CM-Sepharose FF, using the flow-through), hydrophobic interaction chromatography (on Phenyl Sepharose Fast Flow HS), and reverse phase chromatography (on Reverse Phase-Source 30 RPC).
  • the finally purified material was directly applied to capillary without any further desalting step.
  • sample/reference are transferred into PCR vials containing 1/10 volume of neutral marker, and mixed by reverse pipetting, avoiding generating bubbles.
  • bracketing references can be used for each sample as described below.
  • At least three injections of the Standard Reference material are done for the purpose of capillary conditioning.
  • the Standard Reference material is used for comparison of sample data.
  • the migration times MT2 and MT3 at the left and right valleys of -3 and + 3 peaks of the Reference Standard (Start) are determined.
  • the 0 peak is the principal peak of the reference.
  • isoforms between MT2 and MT3 are named “acidic isoforms”. Isoforms with migration times higher than MT3 are named “highly acidic isoforms”. Isoforms with migration times lower than MT2 are named “less acidic isoforms”.
  • the reference and each sample are analyzed by using the functions: manual peak between MT1-M2, MT2-MT3 and MT3-MT4; the manual baseline between 5 and 28 minutes and integration OFF between 0 and MT1 and between MT4 and 30 minutes.
  • % isoform abundance area ⁇ MT ⁇ 1 - MT ⁇ 2 or MT ⁇ 2 - MT ⁇ 3 or MT ⁇ 3 - MT ⁇ 4 Total area ⁇ MT ⁇ 1 - MT ⁇ 4
  • This CZE method was applied to crude harvest samples ( Fig. 9 ) and to purified samples for both perfusion and fed-batch process. Despite some differences observed for the pre-treated harvest samples (e.g. higher proportion of basic isoforms with the perfusion process), these basic isoforms were successfully removed by ion exchange chromatography during the purification process (data not shown). Hence, for the purified product, a comparable isoforms profile was obtained for both processes ( Fig. 10 ).

Claims (9)

  1. Verfahren zur Herstellung von rekombinantem Interleukin-18 Bindungsprotein (IL-18BP) in chinesischen Hamster Ovarzellen (Chinese Hamster Ovary cells, CHO) in einem Bioreaktor unter Serum-freien Kulturbedingungen, wobei das Verfahren ein Fed-Batch Verfahren ist umfassend die Schritte:
    a. Eine Zellvermehrungsphase bei 37°C;
    b. Gegebenenfalls, eine intermediäre Phase bei 33°C;
    c. Eine Produktionsphase bei 29°C.
  2. Verfahren gemäß Anspruch 1, wobei die Gesamtzelldichte in der Produktionsphase zwischen 4 und 8 x 106 Zellen pro mL pro Tag liegt.
  3. Verfahren gemäß Anspruch 2, wobei die Gesamtzelldichte in der Produktionsphase zwischen 4 und 8 x 106 Zellen pro mL pro Tag über mindestens 10 Tage der Zellkultur liegt.
  4. Verfahren gemäß jedem der Ansprüche 1 bis 3, wobei die Viabilität zwischen 100 und 80% liegt.
  5. Verfahren gemäß Anspruch 4, wobei die Viabilität zwischen 100 und 80% über mindestens 10 Tage der Zellkultur liegt.
  6. Verfahren gemäß jedem der Ansprüche 1 bis 5, wobei die Proteinproduktivität höher ist als etwa 150 mg oder etwa 350 mg pro L pro Tag.
  7. Verfahren gemäß jedem der Ansprüche 1 bis 6, weiterhin umfassend den Schritt der Sammlung des Zellkulturüberstandes.
  8. Verfahren gemäß jedem der Ansprüche 1 bis 7, weiterhin umfassend den Schritt der Aufreinigung von IL-18BP.
  9. Verfahren gemäß jedem der Ansprüche 1 bis 8, weiterhin umfassend den Schritt der Formulierung von dem IL-18BP in eine pharmazeutische Zusammensetzung.
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Families Citing this family (24)

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HUP0300423A3 (en) * 2000-02-10 2008-07-28 Abbott Lab Antibodies that bind human interleukin-18 and methods of making and using
BRPI0408246A (pt) * 2003-03-11 2006-03-01 Applied Research Systems vetores de expressão que compreendem o promotor ie2 do mcmv
PL1622939T3 (pl) * 2003-05-13 2012-08-31 Merck Serono Sa Aktywne warianty białka wiążącego IL-18 i ich medyczne zastosowania
PT1675956E (pt) 2003-10-21 2010-11-04 Merck Serono Sa Sequência mínima de adn que actua como um isolador de cromatina e sua utilização na expressão de proteína
CA2544146C (en) * 2003-11-05 2012-10-02 Ares Trading S.A. Process for the purification of il-18 binding protein
US7968684B2 (en) 2003-11-12 2011-06-28 Abbott Laboratories IL-18 binding proteins
JP4713567B2 (ja) 2004-03-01 2011-06-29 アレス トレーディング ソシエテ アノニム 哺乳類細胞のｉｌ−１８ｂｐの産生のための無血清細胞培地の使用
ES2313367T3 (es) 2004-06-29 2009-03-01 Ares Trading S.A. Procedimiento para la purificacion de proteina de union al il-18.
JP5091127B2 (ja) 2005-06-10 2012-12-05 アレス トレーディング ソシエテ アノニム Ｉｌ−１８結合タンパク質の精製のための方法
US20160145589A1 (en) 2011-06-24 2016-05-26 Green Cross Corporation Composition and formulation comprising recombinant human iduronate-2-sulfatase and preparation method thereof
SI2837680T1 (sl) 2011-07-01 2020-07-31 Amgen Inc. Celična kultura sesalca
KR101380740B1 (ko) 2012-06-29 2014-04-11 쉐어 휴먼 제네텍 세러피스, 인코포레이티드 이듀로네이트-2-설파타제의 정제
US9150841B2 (en) 2012-06-29 2015-10-06 Shire Human Genetic Therapies, Inc. Cells for producing recombinant iduronate-2-sulfatase
US20140004097A1 (en) * 2012-06-29 2014-01-02 Shire Human Genetic Therapies, Inc. Method of producing recombinant iduronate-2-sulfatase
US9677105B2 (en) * 2013-03-14 2017-06-13 Momenta Pharmaceuticals, Inc. Methods of cell culture
US8956830B2 (en) 2013-03-14 2015-02-17 Momenta Pharmaceuticals, Inc. Methods of cell culture
US9217168B2 (en) 2013-03-14 2015-12-22 Momenta Pharmaceuticals, Inc. Methods of cell culture
KR20150133833A (ko) * 2013-03-26 2015-11-30 코히러스 바이오사이언시즈, 인코포레이티드 단백질 생산 방법
PT3041864T (pt) * 2013-09-05 2021-10-26 Ab2 Bio Sa Proteína de ligação a il-18 (il-18bp) em doenças inflamatórias
USD759075S1 (en) * 2014-04-11 2016-06-14 Nutonian, Inc. Display screen with graphical user interface
USD759076S1 (en) * 2014-04-18 2016-06-14 Nutonian, Inc. Display screen with graphical user interface
JP7274259B2 (ja) 2015-03-05 2023-05-16 エイビー２ バイオ ソシエテアノニム 炎症性疾患におけるｉｌ－１８結合タンパク質（ｉｌ－１８ｂｐ）および抗体
KR101789509B1 (ko) * 2015-11-05 2017-10-26 주식회사 프로젠 재조합 인간 갑상선 자극 호르몬을 포함하는 조성물 및 상기 재조합 인간 갑상선 자극 호르몬의 생산 방법
CN111212899A (zh) 2017-10-16 2020-05-29 里珍纳龙药品有限公司 灌注生物反应器及相关使用方法

Family Cites Families (37)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4588585A (en) 1982-10-19 1986-05-13 Cetus Corporation Human recombinant cysteine depleted interferon-β muteins
US4737462A (en) 1982-10-19 1988-04-12 Cetus Corporation Structural genes, plasmids and transformed cells for producing cysteine depleted muteins of interferon-β
US4959314A (en) 1984-11-09 1990-09-25 Cetus Corporation Cysteine-depleted muteins of biologically active proteins
US5116943A (en) 1985-01-18 1992-05-26 Cetus Corporation Oxidation-resistant muteins of Il-2 and other protein
US5266476A (en) 1985-06-18 1993-11-30 Yeda Research & Development Co., Ltd. Fibrous matrix for in vitro cell cultivation
US5017691A (en) 1986-07-03 1991-05-21 Schering Corporation Mammalian interleukin-4
US4879111A (en) 1986-04-17 1989-11-07 Cetus Corporation Treatment of infections with lymphokines
US4965195A (en) 1987-10-26 1990-10-23 Immunex Corp. Interleukin-7
US4904584A (en) 1987-12-23 1990-02-27 Genetics Institute, Inc. Site-specific homogeneous modification of polypeptides
DK0567566T4 (da) 1991-01-18 2007-10-22 Amgen Inc Fremgangsmåder til behandling af tumornekrosefaktor-medierede sygdomme
JP4306813B2 (ja) * 1995-09-19 2009-08-05 アスビオファーマ株式会社 動物細胞の新規培養方法
DE69733309T2 (de) 1996-02-20 2006-01-19 Applied Research Systems Ars Holding N.V. Heterodimerebildende hybrid-proteine
DK0912734T3 (da) 1996-07-12 2011-02-07 Genentech Inc Kimæriske heteromultimer-adhæsiner
IL121860A0 (en) 1997-08-14 1998-02-22 Yeda Res & Dev Interleukin-18 binding proteins their preparation and use
WO2001000814A2 (en) 1999-06-25 2001-01-04 Universität Zürich Hetero-associating coiled-coil peptides and screenign method therefor
AU782496B2 (en) 1999-07-13 2005-08-04 Bolder Biotechnology, Inc. Immunoglobulin fusion proteins
IL131047A0 (en) 1999-07-22 2001-01-28 Yeda Res & Dev Use of il-18 inhibitors
UA74557C2 (en) 1999-09-03 2006-01-16 Applied Research Systems A method for producing a heterologous secreted protein from chinese hamster ovaries cells grown on microcarriers
ME00546B (me) 2000-02-21 2011-10-10 Serono Lab Korišćenje inhibitora il-18
KR100798545B1 (ko) 2000-05-05 2008-01-28 어플라이드 리서치 시스템스 에이알에스 홀딩 엔.브이. 죽상경화증의 치료 또는 예방을 위한 ｉｌ-18 저해물질의용도
IL140110A0 (en) * 2000-12-05 2002-02-10 Applied Research Systems Improvement of homogeneity and secretion of recombinant proteins in mammalian systems
WO2002060479A1 (en) 2001-01-29 2002-08-08 Applied Research Systems Ars Holding N.V. Use of il-18 inhibitors for the treatment and/or prevention of heart disease
US7524488B2 (en) 2001-03-08 2009-04-28 Ares Trading S.A. Interleukin-18 mutants
SI1425028T1 (sl) 2001-05-16 2010-02-26 Yeda Res & Dev Uporaba inhibitorjev za il-18 za zdravljenje ali prepreäśevanje sepse
SK288413B6 (sk) 2001-05-25 2016-10-03 Ares Trading S. A. Použitie proteínu viažuceho IL-18
UA78516C2 (en) 2001-08-10 2007-04-10 Applied Research Systems Use of inhibitors of il-18 for treatment and/or prevention of hypersensitivity disorders, and in particular of delayed-type hypersensitivity
SI1487541T1 (sl) 2002-03-22 2009-02-28 Serono Lab Uporaba inhibitorjev il-18 za zdravljenje in /alipreventivo bolezni perifernega oĺ˝ilja
US20070092933A1 (en) 2002-08-14 2007-04-26 Avidis Sa Production of multimeric fusion proteins using a c4bp scaffold
US20050009149A1 (en) 2002-10-11 2005-01-13 West James W. Production of homotrimeric fusion proteins
US7541164B2 (en) * 2002-12-23 2009-06-02 Bristol-Myers Squibb Company Mammalian cell culture processes for protein production
BRPI0408246A (pt) 2003-03-11 2006-03-01 Applied Research Systems vetores de expressão que compreendem o promotor ie2 do mcmv
PL1622939T3 (pl) 2003-05-13 2012-08-31 Merck Serono Sa Aktywne warianty białka wiążącego IL-18 i ich medyczne zastosowania
PT1675956E (pt) 2003-10-21 2010-11-04 Merck Serono Sa Sequência mínima de adn que actua como um isolador de cromatina e sua utilização na expressão de proteína
CA2544146C (en) 2003-11-05 2012-10-02 Ares Trading S.A. Process for the purification of il-18 binding protein
JP4713567B2 (ja) 2004-03-01 2011-06-29 アレス トレーディング ソシエテ アノニム 哺乳類細胞のｉｌ−１８ｂｐの産生のための無血清細胞培地の使用
ES2313367T3 (es) 2004-06-29 2009-03-01 Ares Trading S.A. Procedimiento para la purificacion de proteina de union al il-18.
JP5091127B2 (ja) 2005-06-10 2012-12-05 アレス トレーディング ソシエテ アノニム Ｉｌ−１８結合タンパク質の精製のための方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
CHUPPA SANDRA ET AL: "Fermentor temperature as a tool for control for high-density perfusion cultures of mammalian cells", 1997, BIOTECHNOLOGY AND BIOENGINEERING, VOL. 55, NR. 2, PAGE(S) 328-338, ISSN: 0006-3592 *

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